Methods and compounds for the treatment of immunologically-mediated diseases using mycobacterium vaccae

ABSTRACT

Methods for the prevention and treatment of disorders, including disorders of the respiratory system, such as infection with mycobacteria such as  M. tuberculosis  or  M. avium , sarcoidosis, asthma, allergic rhinitis and lung cancers are provided, such methods comprising administering a composition comprising derivatives of delipidated and deglycolipidated  M vaccae  cells.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Provisional PatentApplication No. 60/137,112, filed Jun. 2, 1999.

TECHNICAL FIELD

The present invention relates generally to methods for the treatment ofimmunologically-mediated disorders. In certain embodiments, theinvention is related to the use of compositions comprising componentsprepared from Micobacterium vaccae, Mycobacterium tuberculosis andMycobacterium smegmatis for the treatment of immunologically-mediateddisorders of the respiratory system, such as sarcoidosis, asthma andlung cancers, for treatment of allergic disorders such as atopicdermatitis, for treatment of diseases that benefit from the reduction ofeosinophilia, for treatment and prevention of infectious diseases, suchas infection with Mycobacterium tuberculosis or Mycobacterium avium, andfor the treatment of atherosclerosis, hypercholesterolemia and otherdisorders that may be improved by modulating IL-10 production.

BACKGROUND OF THE INVENTION

Tuberculosis is a chronic, infectious disease that is caused byinfection with Mycobacterium tuberculosis (M. tuberculosis). It is amajor disease in developing countries, as well as an increasing problemin developed areas of the world, with about 8 million new cases and 3million deaths each year. Although the infection may be asymptomatic fora considerable period of time, the disease is most commonly manifestedas a chronic inflammation of the lungs, resulting in fever andrespiratory symptoms. If left untreated, significant morbidity and deathmay result.

Although tuberculosis can generally be controlled using extendedantibiotic therapy, such treatment is not sufficient to prevent thespread of the disease. Infected individuals may be asymptomatic, butcontagious, for some time. In addition, although compliance with thetreatment regimen is critical, patient behavior is difficult to monitor.Some patients do not complete the course of treatment, which can lead toineffective treatment and the development of drug resistantmycobacteria.

Inhibiting the spread of tuberculosis requires effective vaccination andaccurate, early diagnosis of the disease. Currently, vaccination withlive bacteria is the most efficient method for inducing protectiveimmunity. The most common mycobacterium employed for this purpose isBacille Calmette-Guerin (BCG), an avirulent strain of Mycobacteriumbovis (M. bovis). However, the safety and efficacy of BCG is a source ofcontroversy and some countries, such as the United States, do notvaccinate the general public. Diagnosis of M. tuberculosis infection iscommonly achieved using a skin test, which involves intradermal exposureto tuberculin PPD (protein-purified derivative). Antigen-specific T cellresponses result in measurable induration at the injection site by 48-72hours after injection, thereby indicating exposure to mycobacterialantigens. Sensitivity and specificity have, however, been a problem withthis test, and individuals vaccinated with BCG cannot be distinguishedfrom infected individuals.

A less well-known mycobacterium that has been used for immunotherapy fortuberculosis, and also leprosy, is Microbacterium vaccae (M. vaccae),which is non-pathogenic in humans. However, there is less information onthe efficacy of M. vaccae compared with BCG, and it has not been usedwidely to vaccinate the general public. M. bovis BCG and M. vaccae arebelieved to contain antigenic compounds that are recognized by theimmune system of individuals exposed to infection with M tuberculosis.

Several patents and other publications disclose treatment of variousconditions by administering mycobacteria, including M. vaccae, orcertain mycobacterial fractions. U.S. Pat. No. 4,716,038 disclosesdiagnosis of, vaccination against and treatment of autoimmune diseasesof various types, including arthritic diseases, by administeringmycobacteria, including M. vaccae. U.S. Pat. No. 4,724,144 discloses animmunotherapeutic agent comprising antigenic material derived from M.vaccae for treatment of mycobacterial diseases, especially tuberculosisand leprosy, and as an adjuvant to chemotherapy. International PatentPublication WO 91/01751 discloses the use of antigenic and/orimmunoregulatory material from M. vaccae as an immunoprophylactic todelay and/or prevent the onset of AIDS. International Patent PublicationWO 94/06466 discloses the use of antigenic and/or immunoregulatorymaterial derived from M. vaccae for therapy of HIV infection, with orwithout AIDS and with or without associated tuberculosis.

U.S. Pat. No. 5,599,545 discloses the use of mycobacteria, especiallywhole, inactivated M. vaccae, as an adjuvant for administration withantigens that are not endogenous to M. vaccae. This publicationtheorizes that the beneficial effect as an adjuvant may be due to heatshock protein 65 (hsp65). International Patent Publication WO 92/08484discloses the use of antigenic and/or immunoregulatory material derivedfrom M. vaccae for the treatment of uveitis. International PatentPublication WO 93/16727 discloses the use of antigenic and/orimmunoregulatory material derived from M. vaccae for the treatment ofmental diseases associated with an autoimmune reaction initiated by aninfection. International Patent Publication WO 95/26742 discloses theuse of antigenic and/or immunoregulatory material derived from M. vaccaefor delaying or preventing the growth or spread of tumors. InternationalPatent Publication WO 91/02542 discloses the use of autoclaved M. vaccaein the treatment of chronic inflammatory disorders in which a patientdemonstrates an abnormally high release of IL-6 and/or TNF or in whichthe patient's IgG shows an abnormally high proportion of agalactosylIgG. Among the disorders mentioned in this publication are psoriasis,rheumatoid arthritis, mycobacterial disease, Crohn's disease, primarybiliary cirrhosis, sarcoidosis, ulcerative colitis, systemic lupuserythematosus, multiple sclerosis, Guillain-Barre syndrome, primarydiabetes mellitus, and some aspects of graft rejection.

M. vaccae is apparently unique among known mycobacterial species in thatheat-killed preparations retain vaccine and immunotherapeuticproperties. For example, M. bovis BCG vaccines, used for vaccinationagainst tuberculosis, employ live strains. Heat-killed M. bovis BCG andM. tuberculosis have no protective properties when employed in vaccines.A number of compounds have been isolated from a range of mycobacterialspecies that have adjuvant properties. The effect of such adjuvants isessentially to stimulate a particular immune response mechanism againstan antigen from another species.

There are two general classes of compounds that have been isolated frommycobacterial species that exhibit adjuvant properties. The first arewater soluble wax D fractions (White et al., Immunology 1:54, 1958; U.S.Pat. No. 4,036,953). The second are muramyl dipeptide-based substances(N-acetyl glucosamine and N-glycolymuramic acid in approximatelyequimolar amounts) as described in U.S. Pat. Nos. 3,956,481 and4,036,953. These compounds differ from the delipidated anddeglycolipidated M. vaccae (DD-M. vaccae) of the present invention inthe following aspects of their composition:

1. They are water-soluble agents, whereas DD-M. vaccae is insoluble inaqueous solutions.

2. They consist of a range of small oligomers of the mycobacterial cellwall unit, either extracted from bacteria by various solvents, ordigested from the cell wall by an enzyme. In contrast, DD-M. vaccaecomprises processed mycobacterial cells.

3. All protein has been removed from their preparations by digestionwith proteolytic enzymes. The only constituents of their preparationsare the components of the cell wall peptidoglycan structure, namelyalanine, glutamic acid, diaminopimelic acid, N-acetyl glucosamine, andN-glycolylmuramic acid. In contrast, DD-M. vaccae contains 50% w/wprotein, comprising a number of distinct protein species.

Sarcoidosis is a disease of unknown cause characterized by granulomatousinflammation affecting many organs of the body and especially the lungs,lymph nodes and liver. Sarcoid granulomata are composed of mononuclearphagocytes, with epithelioid and giant cells in their center, and Tlymphocytes. CD4 T lymphocytes are closely associated with theepithelioid cells while both CD4 and CD8 T lymphocytes accumulate at theperiphery. The characteristic immunological abnormalities in sarcoidosisinclude peripheral blood and bronchoalveolar lavage hyper-globulinaemiaand depression of ‘delayed type’ hypersensitivity reactions in the skinto tuberculin and other similar antigens, such as Candida and mumps.Peripheral blood lymphocyte numbers are reduced and CD4:CD8 ratios inperipheral blood are depressed to approximately 1-1.5:1. These are notmanifestations of a generalized immune defect, but rather theconsequence of heightened immunological activity which is‘compartmentalized’ to sites of disease activity. In patients withpulmonary sarcoidosis, the total number of cells recovered bybronchoalveolar lavage is increased five-to ten-fold and the proportionof lymphocytes increased from the normal of less than 10-14% to between15% and 50%. More than 90% of the lymphocytes recovered are Tlymphocytes and the CD4:CD8 ratio has been reported to be increased fromthe value of 1.8:1 in normal controls to 10.5:1. The T lymphocytes arepredominantly of the Th1 class, producing IFN-γ and IL-2 cytokines,rather than of the Th2 class. Following treatment, the increase in Th1lymphocytes in sarcoid lungs is corrected.

Sarcoidosis involves the lungs in nearly all cases. Even when lesionsare predominantly seen in other organs, subclinical lung involvement isusually present. While some cases of sarcoidosis resolve spontaneously,approximately 50% of patients have at least a mild degree of permanentorgan dysfunction. In severe cases, lung fibrosis develops andprogresses to pulmonary failure requiring lung transplantation. Themainstay of treatment for sarcoidosis is corticosteroids. Patientsinitially responding to corticosteroids often relapse and requiretreatment with other immunosuppressive drugs such as methotrexate orcyclosporine.

Asthma is a common disease, with a high prevalence in the developedworld. Asthma is characterized by increased responsiveness of thetracheobronchial tree to a variety of stimuli, the primary physiologicaldisturbance being reversible airflow limitation, which may bespontaneous or drug-related, and the pathological hallmark beinginflammation of the airways. Clinically, asthma can be subdivided intoextrinsic and intrinsic variants.

Extrinsic asthma has an identifiable precipitant, and can be thought ofas being atopic, occupational and drug-induced. Atopic asthma isassociated with the enhancement of a Th2-type of immune response withthe production of specific immunoglobulin E (IgE), positive skin teststo common aeroallergens and/or atopic symptoms. It can be dividedfurther into seasonal and perennial forms according to the seasonaltiming of symptoms. The airflow obstruction in extrinsic asthma is dueto nonspecific bronchial hyperesponsiveness caused by inflammation ofthe airways. This inflammation is mediated by chemicals released by avariety of inflammatory cells including mast cells, eosinophils andlymphocytes. The actions of these mediators result in vascularpermeability, mucus secretion and bronchial smooth muscle constriction.In atopic asthma, the immune response producing airway inflammation isbrought about by the Th2 class of T cells which secrete IL-4, IL-5 andIL-10. It has been shown that lymphocytes from the lungs of atopicasthmatics produce IL-4 and IL-5 when activated. Both IL-4 and IL-5 arecytokines of the Th2 class and are required for the production of IgEand involvement of eosimophils in asthma. Occupational asthma may berelated to the development of IgE to a protein hapten, such as acidanhydrides in plastic workers and plicatic acid in some western redcedar-induced asthma, or to non-IgE related mechanisms, such as thatseen in toluene diiusocyanate-induced asthma. Drug-induced asthma can beseen after the administration of aspirin or other non-steroidalanti-inflammatory drugs, most often in a certain subset of patients whomay display other features such as nasal polyposis and sinusitis.Intrinsic or cryptogenic asthma is reported to develop after upperrespiratory tract infections, but can arise de novo in middle-aged orolder people, in whom it is more difficult to treat than extrinsicasthma.

Asthma is ideally prevented by the avoidance of triggering allergens butthis is not always possible nor are triggering allergens always easilyidentified. The medical therapy of asthma is based on the use ofcorticosteroids and bronchodilator drugs to reduce inflammation andreverse airway obstruction. In chronic asthma, treatment withcorticosteroids leads to unacceptable adverse side effects.

Another disorder with a similar immune abnormality to asthma is allergicrhinitis. Allergic rhinitis is a common disorder and is estimated toaffect at least 10% of the population. Allergic rhinitis may be seasonal(hay fever) caused by allergy to pollen. Non-seasonal or perennialrhinitis is caused by allergy to antigens such as those from house dustmite or animal dander.

The abnormal immune response in allergic rhinitis is characterized bythe excess production of IgE antibodies specific against the allergen.The inflammatory response occurs in the nasal mucosa rather than furtherdown the airways as in asthma. Like asthma, local eosinophilia in theaffected tissues is a major feature of allergic rhinitis. As a result ofthis inflammation, patients develop sneezing, nasal discharge andcongestion. In more severe cases, the inflammation extends to the eyes(conjunctivitis), palate and the external ear. While it is not lifethreatening, allergic rhinitis may be very disabling, prevent normalactivities, and interfere with a person's ability to work. Currenttreatment involves the use of antihistamines, nasal decongestants and,as for asthma, sodium cromoglycate and corticosteroids.

Atopic dermatitis is a chronic pruritic inflammatory skin disease whichusually occurs in families with an hereditary predisposition for variousallergic disorders, such as allergic rhinitis and asthma. Atopicdermatitis occurs in approximately 10% of the general population. Themain symptoms are dry skin, dermatitis (eczema) localised mainly in theface, neck and on the flexor sides and folds of the extremitiesaccompanied by severe itching. It typically starts within the first twoyears of life. In about 90% of the patients this skin disease disappearsduring childhood but the symptoms can continue into adult life. It isone of the commonest forms of dermatitis world-wide. It is generallyaccepted that in atopy and in atopic dermatitis, a T cell abnormality isprimary and that the dysfunction of T cells which normally regulate theproduction of IgE is responsible for the excessive production of thisimmunoglobulin.

Allergic contact dermatitis is a common non-infectious inflammatorydisorder of the skin. In contact dermatitis, immunological reactionscannot develop until the body has become sensitised to a particularantigen. Subsequent exposure of the skin to the antigen and therecognition of these antigens by T cells result in the release ofvarious cytokines, proliferation and recruitment of T cells and finallyin dermatitis (eczema).

Only a small proportion of the T cells in a lesion of allergic contactdermatitis are specific for the relevant antigen. Activated T cellsprobably migrate to the sites of inflammation regardless ofantigen-specificity. Delayed-type hypersensitivity can only betransferred by T cells (CD4⁺ cells) sharing the MHC class II antigens.The ‘response’ to contact allergens can be transferred by T cellssharing either MHC class I (CD8⁺ cells) or class II (CD4⁺ cells)molecules (Sunday, M. E. et al., J. Immunol. 125:1601-1605, 1980).Keratinocytes can produce interleukin-1 which can facilitate the antigenpresentation to T cells. The expression of the surface antigenintercellular adhesion molecule-1 (ICAM-1) is induced both onkeratinocytes and endothelium by the cytokines tumor necrosis factor(TNF) and interferon-gamma (IFN-γ).

If the causes can be identified, removal alone will cure allergiccontact dermatitis. During active inflammation, topical corticosteroidsare useful. An inhibitory effect of cyclosporin has been observed indelayed-type hypersensitivity on the pro-inflammatory function(s) ofprimed T cells in vitro (Shidani, B. et al., Eur. J. Immunol.14:314-318, 1984). The inhibitory effect of cyclosporin on the earlyphase of T cell activation in mice has also been reported (Milon, G. etal., Ann. Immunol. (Inst. Pasteur) 135d:237-245, 1984).

Lung cancer is the leading cause of death from cancer. The incidence oflung cancer continues to rise and the World Health Organizationestimates that by 2000AD there will be 2 million new cases annually.Lung cancers may be broadly classified into two categories: small celllung cancer (SCLC) which represents 20-25% of all lung cancers, andnon-small cell lung cancer (NSCLC) which accounts for the remaining 75%.The majority of SCLC is caused by tobacco smoke. SCLC tends to spreadearly and 90% of patients present at diagnosis with involvement of themediastinal lymph nodes in the chest. SCLC is treated by chemotherapy,or a combination of chemotherapy and radiotherapy. Complete responserates vary from 10% to 50%. For the rare patient without lymph nodeinvolvement, surgery followed by chemotherapy may result in cure ratesexceeding 60%. The prognosis for NSCLC is more dismal, as most patientshave advanced disease by the time of diagnosis. Surgical removal of thetumor is possible in a very small number of patients and the five yearsurvival rate for NSCLC is only 5-10%.

The factors leading to the development of lung cancer are complex andmultiple. Environmental and genetic factors interact and causesequential and incremental abnormalities that lead to uncontrolled cellproliferation, invasion of adjacent tissues and spread to distant sites.Both cell-mediated and humoral immunity have been shown to be impairedin patients with lung cancer. Radiotherapy and chemotherapy furtherimpair the immune function of patients. Attempts have been made toimmunize patients with inactivated tumor cells or tumor antigens toenhance host anti-tumor response. Bacille Calmette-Guerin (BCG) has beenadministered into the chest cavity following lung cancer surgery toaugment non-specific immunity. Attempts have been made to enhanceanti-tumor immunity by giving patients lymphocytes treated ex vivo withinterleukin-2 (IL-2). These lymphokine-activated lymphocytes acquire theability to kill tumor cells. Current immunotherapies for lung cancer arestill at a developmental stage and their efficacies have yet to beestablished for the standard management of lung cancer.

Atherosclerosis is a chronic inflammatory disease of the arterial wallthat is characterized by accumulation of lipids, macrophages, Tlymphocytes, smooth muscle cells and extracellular matrix.Anti-inflammatory cytokines are produced during the inflammatoryreaction and are believed to modulate the inflammatory process.Interleukin-10 (IL-10) is secreted by Th2 lymphocytes and bymacrophages, and is known to have anti-inflammatory properties. Mallatet al. recently reported studies in which IL-10 was shown to have aprotective effect in the formation and stability of atheroscleroticlesions in mice (Circ. Res. 1999, 85:el7-24). These studies suggest thatagents that increase IL-10 production may be employed to modulate theextent and/or severity of atherosclerosis.

Other disorders in which administration of IL-10 has been shown tobeneficial include hypercholesterolemia (see U.S. Pat. No. 5,945,097);bacterial infections, including infection with gram-negative and/orgram-positive bacteria (see U.S. Pat. Nos. 5,837,293 and 5,837,232); andinsulin-dependent diabetes mellitus (see, U.S. Pat. No. 5,827,513). Inaddition, U.S. Pat. No. 5,871,725 discloses a method of treating cancerby administering to a patient peripheral blood mononuclear cells (PBMC)that have been activated with IL-10.

SUMMARY OF THE INVENTION

Briefly stated, the present invention provides compositions and methodsfor the prevention and treatment of immunologically-mediated disorders,including disorders of the respiratory system (such as infection withmycobacteria such as M. tuberculosis or M. avium, sarcoidosis, asthma,allergic rhinitis and lung cancers), allergic disorders such as atopicdermatitis, diseases that benefit from the reduction of eosinophilia,and disorders that may be improved by modulating IL-10 production (suchas atherosclerosis, hypercholesterolemia, cancer, bacterial infectionsand insulin-dependent diabetes mellitus).

In a first aspect, compositions comprising delipidated anddeglycolipidated mycobacterial cells are provided. In specificembodiments, the delipidated and deglycolipidated cells are preparedfrom M. vaccae, M. tuberculosis and/or M. smegmatis.

In a second aspect, the present invention provides compositionscomprising a derivative of delipidated and deglycolipidatedmycobacterial cells, the derivative of delipidated and deglycolipidatedmycobacterial cells being selected from the group consisting of:delipidated and deglycolipidated mycobacterial cells that have beentreated by alkaline hydrolysis; delipidated and deglycolipidatedmycobacterial cells that have been treated by acid hydrolysis; anddelipidated and deglycolipidated mycobacterial cells that have beentreated with periodic acid. In preferred embodiments, such compositionscomprise a derivative of delipidated and deglycolipidated M. vaccaecells, the derivative of delipidated and deglycolipidated M. vaccaecells being selected from the group consisting of: delipidated anddeglycolipidated M. vaccae cells that have been treated by alkalinehydrolysis; delipidated and deglycolipidated M. vaccae cells that havebeen treated by acid hydrolysis; and delipidated and deglycolipidated M.vaccae cells that have been treated with periodic acid. The derivativesof delipidated and deglycolipidated M. vaccae preferably containgalactose in an amount less than 9.7% of total carbohydrate, morepreferably less than 5% of total carbohydrate, and most preferably lessthan 3.5% total carbohydrate. In certain embodiments, the derivatives ofdelipidated and deglycolipidated M. vaccae contain glucosamine in anamount greater than 3.7% of total carbohydrate, preferably greater than5% total carbohydrate and more preferably greater than 7.5% totalcarbohydrate.

In further aspects of this invention, methods are provided for thetreatment of a disorder in a patient, including disorders of therespiratory system, such methods comprising administering to the patienta composition of the present invention. In certain embodiments, thedisorder is selected from the group consisting of mycobacterialinfections, asthma, sarcoidosis, allergic rhinitis and lung cancers. Inone embodiment, the compositions are administered to the airways leadingto or located within the lungs, preferably by inhalation through thenose or mouth, and are preferably administered in aerosol forms. Thecompositions may also, or alternatively, be administered by intradermal,transdermal or subcutaneous routes.

In another aspect, the present invention provides methods for thetreatment of a disorder of the respiratory system in a patient by theadministration of a composition of the present invention, wherein thedisorder is characterized by the presence of eosinophilia in the tissuesof the respiratory system. Examples of such diseases include asthma andallergic rhinitis. In a related aspect, the present invention providesmethods for the reduction of eosinophilia in a patient, such methodscomprising administering at least one of the compositions disclosedherein. Typically, the reduction in eosinophilia will vary between about20% and about 80%. The percentage of reduction in lung eosinophilia canbe determined by measuring the number of eosinophils in bronchoalveolarlavage fluid before and after treatment as described below.

In yet a further aspect, methods for enhancing the production of IL-10are provided, such methods comprising administering a composition of thepresent invention. As discussed above, it has recently been shown thatIL-10 plays a protective role in the formation and stability ofatherosclerotic lesions. IL-10 has also been shown to be effective inthe treatment of hypercholesterolemia, cancer, bacterial infections, andinsulin-dependent diabetes mellitus. The inventive compositions may thusbe usefully employed in the treatment of such disorders.

The present invention further provides methods for the activation of γδT cells, γδ T cells or NK cells, and thereby repairing epithelium, in apatient, comprising administering to the patient a composition of thepresent invention.

These and other aspects of the present invention will become apparentupon reference to the following detailed description and attacheddrawings. All references disclosed herein are hereby incorporated byreference in their entirety as if each was incorporated individually.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the induction of IL-12 by autoclaved M. vaccae,lyophilized M. vaccae, delipidated and deglycolipidated M. vaccae, andM. vaccae glycolipids.

FIG. 2 compares the in vitro stimulation of interferon-gamma productionin spleen cells from Severe Combined ImmunoDeficient (SCID) mice bydifferent concentrations of heat-killed (autoclaved) M. vaccae,delipidated and deglycolipidated M. vaccae, and M. vaccae glycolipids.

FIG. 3 shows the suppression by DD-M. vaccae (Q₁) and the DD-M. vaccaederivatives Q2 (DD-M. vaccae-KOH), Q3 (DD-M. vaccae-acid), Q4 (DD-M.vaccae-periodate), Q6 (DD-M. vaccae-KOH-periodate), P5 (DD-M.vaccae-KOH-acid) and P6 (DD-M. vaccae-KOH-periodate) ofovalbumin-induced airway eosinophilia in mice vaccinated intranasallywith these compounds. Control mice received PBS.

FIG. 4 shows the stimulation of IL-10 production in THP-1 cells byderivatives of DD-M. vaccae.

FIG. 5 illustrates the effect of immunizing mice with heat-killed M.vaccae or delipidated and deglycolipidated M. vaccae (DD-M. vaccae )prior to infection with tuberculosis.

FIG. 6 illustrates the re-suspension of DD-M. vaccae and DD-M.vaccae-KOH.

FIG. 7 shows the stimulation of IL-12 production in macrophages by DD-M.vaccae (R1) and the DD-M. vaccae derivatives DD-M. vaccae-KOH (R2),(DD-M. vaccae-acid (R3), DD-M. vaccae-periodate (R4), DD-M.vaccae-KOH-acid (R5) and DD-M. vaccae-KOH-periodate (R6).

FIG. 8 illustrates the suppression of airway eosinophilia in adose-dependent manner by a DD-M. vaccae-acid derivative.

FIG. 9 compares the effect of intranasal and intradermal immunizationwith DD-M. vaccae-acid on the suppression of lung eosinophils.

FIG. 10 illustrates the effect of immunization with DD-M. vaccae, DD-M.tuberculosis and DD-M. smegmatis on airway eosinophilia.

FIG. 11 illustrates TNF-α production by human PBMC and non-adherentcells stimulated with DD-M. vaccae.

FIGS. 12A and 12B illustrate IL-10 and IFN-γ production, respectively,by human PBMC and non-adherent cells stimulated with DD-M. vaccae.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, the present invention is generally directed tocompositions and methods for the treatment of immunologically-mediateddisorders. In certain specific embodiments, such disorders are selectedfrom the group consisting of disorders of the respiratory system,allergic disorders and disorders in which administration of IL-10 and/orstimulation of IL-10 production are beneficial. Examples of respiratorysystem disorders include mycobacterial infection, asthma, sarcoidosis,allergic rhinitis and lung cancer. Examples of disorders in whichadministration and/or increased production of IL-10 are believed to bebeneficial include atherosclerosis, hypercholesterolemia, cancer,bacterial infections, and insulin-dependent diabetes mellitus.

Certain pathogens, such as M. tuberculosis, as well as certain cancers,are effectively contained by an immune attack directed by CD4⁺ T cells,known as cell-mediated immunity. Other pathogens, such as poliovirus,also require antibodies, produced by B cells, for containment. Thesedifferent classes of immune attack (T cell or B cell) are controlled bydifferent subpopulations of CD4⁺ T cells, commonly referred to as Th1and Th2 cells.

The two types of Th cell subsets have been well characterized in amurine model and are defined by the cytokines they release uponactivation. The Th1 subset secretes IL-2, IFN-γ and tumor necrosisfactor, and mediates macrophage activation and delayed-typehypersensitivity response. The Th2 subset releases IL-4, IL-5, IL-6 andIL-10, which stimulate B cell activation. The Th1 and Th2 subsets aremutually inhibiting, so that IL-4 inhibits Th1-type responses, and IFN-γinhibits Th2-type responses. Similar Th1 and Th2 subsets have been foundin humans, with release of the identical cytokines observed in themurine model. Amplification of Th1-type immune responses is central to areversal of disease state in many disorders, including disorders of therespiratory system such as tuberculosis, sarcoidosis, asthma, allergicrhinitis and lung cancers. IL-12 has been shown to up-regulate Th1responses, while IL-10 has been shown to down-regulate Th2 responses.Zuany-Amorim et al. have shown that IL-10 regulates leukocyteinfiltration into the airways of antigen-challenged mice, indicatingthat IL-10 plays an important role in regulating allergic inflammatoryprocesses in the lung. (J. Clin. Invest. 1995, 95:2644-2651). Studies byBorish et al. have found that bronchoalveolar fluid from asthmaticpatients contains reduced levels of IL-10 compared to that from normaldonors (J. Allergy Clin. Immunol. 1996, 97:1288-96).

In one aspect, methods are provided for the treatment of respiratoryand/or lung disorders, comprising administering delipidated anddeglycolipidated mycobacterial cells, preferably delipidated anddeglycolipidated M. tuberculosis cells and/or delipidated anddeglycolipidated M. smegmatis cells. In a related aspect, the presentinvention provides methods for the immunotherapy of respiratory and/orlung disorders, including tuberculosis, sarcoidosis, asthma, allergicrhinitis and lung cancers, in a patient by administration of acomposition that comprises at least one derivative of delipidated anddeglycolipidated mycobacterial cells. In certain specific embodiments,such methods comprise administering at least one derivative of DD-M.vaccae. As detailed below, the inventors have demonstrated thatadministration of such compositions is effective in the treatment ofasthma in a mouse model. These compositions are believed to be effectivein the treatment of diseases such as asthma due to their ability tosuppress asthma-inducing Th2 immune responses. In one embodiment, thecompositions are delivered directly to the mucosal surfaces of airwaysleading to and/or within the lungs. However, the compositions may also,or alternatively, be administered via intradermal or subcutaneousroutes.

As used herein the term “respiratory system” refers to the lungs, nasalpassageways, trachea and bronchial passageways.

As used herein the term “airways leading to or located in the lung”includes the nasal passageways, mouth, tonsil tissue, trachea andbronchial passageways.

As used herein, a “patient” refers to any warm-blooded animal,preferably a human. Such a patient may be afflicted with disease or maybe free of detectable disease. In other words, the inventive methods maybe employed to induce protective immunity for the prevention ortreatment of disease.

As used herein the term “inactivated M. vaccae” refers to M. vaccaecells that have either been killed by means of heat, as detailed belowin Example 1, or subjected to radiation, such as ⁶⁰Cobalt at a dose of2.5 megarads. As used herein, the term “modified M. vaccae” includesdelipidated M. vaccae cells, deglycolipidated M. vaccae cells, M. vaccaecells that have been both delipidated and deglycolipidated (DD-M.vaccae), and derivatives of delipidated and deglycolipidated M. vaccaecells. DD-M. vaccae may be prepared as described below in Example 1,with the preparation of derivatives of DD-M. vaccae being detailed belowin Example 2. The preparation of delipidated and deglycolipidated M.tuberculosis (DD-M. tuberculosis) and M. smegmatis (DD-M. smegmatis) isdescribed in Example 10 below. Derivatives of DD-M. tuberculosis andDD-M. smegmatis, such as acid-treated, alkali-treated and/orperiodate-treated derivatives, may be prepared using the proceduresdisclosed herein for the preparation of derivatives of DD-M. vaccae.

The derivatives of DD-M. vaccae preferably contain galactose in anamount less than 9.7% of total carbohydrate, more preferably less than5% of total carbohydrate, and most preferably less than 3.5% totalcarbohydrate. In certain embodiments, the derivatives of DD-M. vaccaepreferably contain glucosamine in an amount greater than 3.7% of totalcarbohydrate, more preferably greater than 5% total carbohydrate, andmost preferably greater than 7.5% total carbohydrate. Derivativesprepared by treatment of DD-M. vaccae with alkali, such as DD-M.vaccae-KOH have a reduced number of ester bonds linking mycolic acids tothe arabinogalactan of the cell wall compared to DD-M. vaccae.Derivatives prepared by treatment with acid, such as DD-M. vaccae-acid,have a reduced number of phosphodiester bonds attaching arabinogalactansidechains to the peptidoglycan of the cell wall. Derivatives preparedby treatment of DD-M. vaccae with periodate, such as DD-M.vaccae-periodate, have a reduced number of cis-diol-containing sugarresidues compared to DD-M. vaccae.

In general, the inventive compositions may be administered by injection(e.g., intradermal, intramuscular, intravenous or subcutaneous),intranasally (e.g., by aspiration), orally or epicutaneously (appliedtopically onto skin). In one embodiment, the compositions of the presentinvention are in a form suitable for delivery to the mucosal surfaces ofthe airways leading to or within the lungs. For example, the compositionmay be suspended in a liquid formulation for delivery to a patient in anaerosol form or by means of a nebulizer device similar to thosecurrently employed in the treatment of asthma.

For use in therapeutic methods, the inventive compositions mayadditionally contain a physiologically acceptable carrier and/or animmunostimulant that elicits and/or stimulates an immune response, suchas an adjuvant or a liposome, into which the polypeptide isincorporated. While any suitable carrier known to those of ordinaryskill in the art may be employed in the pharmaceutical compositions ofthis invention, the type of carrier will vary depending on the mode ofadministration. For parenteral administration, such as subcutaneousinjection, the carrier preferably comprises water, saline, alcohol, afat, a wax or a buffer. For oral administration, any of the abovecarriers or a solid carrier, such as mannitol, lactose, starch,magnesium stearate, sodium saccharine, talcum, cellulose, glucose,sucrose, and magnesium carbonate, may be employed. Biodegradablemicrospheres (e.g., polylactic galactide) may also be employed ascarriers for the pharmaceutical compositions of this invention. Suitablebiodegradable microspheres are disclosed, for example, in U.S. Pat. No.Nos. 4,897,268 and 5,075,109.

Any of a variety of adjuvants may be employed in the compositions ofthis invention to non-specifically enhance the immune response. Mostadjuvants contain a substance designed to protect the antigen from rapidcatabolism, such as aluminum hydroxide or mineral oil, and anon-specific stimulator of immune responses, such as lipid A, Bordetellapertussis, M. tuberculosis, or, as discussed below, M. vaccae. Suitableadjuvants are commercially available as, for example, Freund'sIncomplete Adjuvant and Freund's Complete Adjuvant (Difco Laboratories,Detroit, Mich.), and Merck Adjuvant 65 (Merck and Company, Inc., Rahway,N.J.). Other suitable adjuvants include alum, biodegradablemicrospheres, monophosphoryl lipid A and Quil A.

The preferred frequency of administration and effective dosage will varyfrom individual to individual. For both DD-M. vaccae and derivatives ofDD-M. vaccae, the amount present in a dose preferably ranges from about10 μg to about 1000 μg, more preferably from about 10 μg to about 100μg. The number of doses may range from 1 to about 10 administered over aperiod of up to 12 months.

The word “about,” when used in this application with reference to theamount of active component in a dose, contemplates a variance of up to5% from the stated amount. The word “about,” when used with reference toa percentage reduction of eosinophils, contemplates a variance of up to10% from the stated percentage.

The following examples are offered by way of illustration and are notlimiting.

EXAMPLE 1 Preparation of Delipidated and Deglycolipidated M. vaccaeCells (DD-M. vaccae)

This example illustrates the processing of different constituents of M.vaccae and their immune modulating properties.

Heat-killed M. vaccae and M. vaccae culture filtrate

M. vaccae (ATCC Number 15483) was cultured in sterile Medium 90 (yeastextract, 2.5 g/l; tryptone, 5 g/l; glucose 1 g/l) at 37° C. The cellswere harvested by centrifugation, and transferred into sterileMiddlebrook 7H9 medium (Difco Laboratories, Detroit, Mich.) with glucoseat 37° C. for one day. The medium was then centrifuged to pellet thebacteria, and the culture filtrate removed. The bacterial pellet wasresuspended in phosphate buffered saline at a concentration of 10 mg/ml,equivalent to 10¹⁰ M. vaccae organisms per ml. The cell suspension wasthen autoclaved for 15 min at 120° C. The culture filtrate was passagedthrough a 0.45 μm filter into sterile bottles.

Preparation of Delipidated and Deglycolipidated M. vaccae (DD-M. vaccae)and Compositional Analysis

To prepare delipidated M. vaccae, the autoclaved M. vaccae was pelletedby centrifugation, the pellet washed with water and collected again bycentrifugation, and freeze-dried. An aliquot of this freeze-dried M.vaccae was set aside and referred to as lyophilised M. vaccae. When usedin experiments it was resuspended in PBS to the desired concentration.Freeze-dried M. vaccae was treated with chloroform/methanol (2:1) for 60mins at room temperature to extract lipids, and the extraction wasrepeated once. The delipidated residue from the chloroform/methanolextraction was further treated with 50% ethanol to remove glycolipids byrefluxing for two hours. The 50% ethanol extraction was repeated twotimes. The pooled 50% ethanol extracts were used as a source of M.vaccae glycolipids (see below). The residue from the 50% ethanolextraction was freeze-dried and weighed. The amount of delipidated anddeglycolipidated M. vaccae prepared was equivalent to 11.1% of thestarting wet weight of M. vaccae used. For bioassay, the delipidated anddeglycolipidated M. vaccae (DD-M. vaccae), was resuspended inphosphate-buffered saline by sonication, and sterilised by autoclaving.

The compositional analyses of heat-killed M. vaccae and DD-M. vaccae arepresented in Table 1. Major changes are seen in the fatty acidcomposition and amino acid composition of DD-M. vaccae as compared tothe insoluble fraction of heat-killed M. vaccae. The data presented inTable 1 show that the insoluble fraction of heat-killed M. vaccaecontains 10% w/w of lipid, and the total amino acid content is 2750nmoles/mg, or approximately 33% w/w. DD-M.vaccae contains 1.3% w/w oflipid and 4250 nmoles/mg amino acids, which is approximately 51% w/w.

TABLE 1 Compositional analyses of heat-killed M. vaccae and DD-M. vaccaeMONOSACCHARIDE COMPOSITION sugar alditol M. vaccae DD-M. vaccae Inositol3.2% 1.7% Ribitol* 1.7% 0.4% Arabinitol 22.7% 27.0% Mannitol 8.3% 3.3%Galactitol 11.5% 12.6% Glucitol 52.7% 55.2%

FATTY ACID COMPOSITION Fatty acid M. vaccae DD-M. vaccae C14:0 3.9%10.0% C16:0 21.1% 7.3% C16:1 14.0% 3.3% C18:0 4.0% 1.5% C18:1* 1.2% 2.7%C18:1w9 20.6% 3.1% C18:1w7 12.5% 5.9% C22:0 12.1% 43.0% C24:1* 6.5%22.9%

The insoluble fraction of heat-killed M. vaccae contains 10% w/w oflipid, and DD-M. vaccae contains 1.3% w/w of lipid.

AMINO ACID COMPOSITION nmoles/mg M. vaccae DD-M. vaccae ASP 231 361 THR170 266 SER 131 199 GLU 319 505 PRO 216 262 GLY 263 404 ALA 416 621 CYS*24 26 VAL 172 272 MET* 72 94 ILE 104 171 LEU 209 340 TYR 39 75 PHE 76132 GlcNH2 5 6 HIS 44 77 LYS 108 167 ARG 147 272

The total amino acid content of the insoluble fraction of heat-killed M.vaccae is 2750 nmoles/mg, or approximately 33% w/w. The total amino acidcontent of DD-M. vaccae is 4250 nmoles/mg, or approximately 51% w/w.

M. vaccae glycolipids

The pooled 50% ethanol extracts described above were dried by rotaryevaporation, redissolved in water, and freeze-dried. The amount ofglycolipid recovered was 1.2% of the starting wet weight of M. vaccaeused. For bioassay, the glycolipids were dissolved in phosphate-bufferedsaline.

Production of Interleukin-12 from Macrophages

Whole heat-killed M. vaccae and DD-M. vaccae were shown to havedifferent cytokine stimulation properties. The stimulation of a Th1immune response is enhanced by the production of interleukin-12 (IL-12)from macrophages. The ability of different M. vaccae preparations tostimulate IL-12 production was demonstrated as follows.

A group of C57BL/6J mice were injected intraperitoneally with DIFCOthioglycolate. After three days peritoneal macrophages were collectedand placed in cell culture with interferon-gamma for three hours. Theculture medium was replaced and various concentrations of wholeheat-killed (autoclaved) M. vaccae, lyophilized M. vaccae, DD-M. vaccae(referred to as delipidated-deglycolipidated M. vaccae in FIG. 1) and M.vaccae glycolipids were added. After a further three days at 37° C., theculture supernatants were assayed for the presence of IL-12 produced bymacrophages. As shown in FIG. 1, the M. vaccae preparations stimulatedthe production of IL-12 from macrophages.

By contrast, these same M. vaccae preparations were examined for theability to stimulate interferon-gamma (IFN-γ) production from NaturalKiller (NK) cells. Spleen cells were prepared from Severe CombinedImmunodeficient (SCID) mice. These populations contain 75-80% NK cells.The spleen cells were incubated at 37° C. in culture with differentconcentrations of heat-killed M. vaccae, DD-M. vaccae, or M. vaccaeglycolipids. The data shown in FIG. 2 demonstrates that, whileheat-killed M. vaccae and M. vaccae glycolipids stimulate production ofinterferon-gamma, DD-M. vaccae stimulated relatively lessinterferon-gamma. The combined data from FIGS. 1 and 2 indicate that,compared with whole heat-killed M. vaccae, DD-M. vaccae is a betterstimulator of IL-12 than of interferon-gamma.

EXAMPLE 2 Preparation and Characterisation of Additional Derivates M.vaccae

Alkaline Hydrolysis of DD-M. vaccae

This procedure is intended to cleave linkages that are labile toalkaline lysis, such as the ester bonds linking mycolic acids to thearabinogalactan of the mycobacterial cell wall.

One gram of DD-M. vaccae, prepared as described in Example 1, wassuspended in 20 ml of a 0.5% solution of potassium hydroxide (KOH) inethanol. Other alkaline agents and solvents are well known in the artand may be used in the place of KOH and ethanol. The mixture wasincubated at 37° C. with intermittent mixing for 48 hours. The solidresidue was harvested by centrifugation, and washed twice with ethanoland once with diethyl ether. The product was air-dried overnight. Theyield was 1.01 g (101%) of KOH-treated DD-M. vaccae, subsequentlyreferred to as DD-M. vaccae-KOH. This derivative was found to be moresoluble than the other derivatives of DD-M. vaccae disclosed herein.

Acid Hydrolysis of DD-M. vaccae

This procedure is intended to cleave acid-labile linkages, such as thephosphodiester bonds attaching the arabinogalactan sidechains to thepeptidoglycan of the mycobacterial cell wall.

DD-M. vaccae or DD-M. vaccae-KOH (100 mg) was washed twice in 1 ml of 50mM H₂SO₄ followed by resuspension and centrifugation. Other acids arewell known in the art and may be used in place of sulphuric acid. Forthe acid hydrolysis step, the solid residue was resuspended in 1 ml of50 mM H₂SO₄, and incubated at 60° C. for 72 hours. Following recovery ofthe solid residue by centrifugation, the acid was removed by washing theresidue five times with water. The freeze-dried solid residue yielded58.2 mg acid-treated DD-M. vaccae (DD-M. vaccae-acid) or 36.7 mgacid-treated DD-M. vaccae-KOH (DD-M. vaccae-KOH-acid).

Periodic Acid Cleavage of DD-M. vaccae

This procedure is intended to cleave cis-diol-containing sugar residuesin DD-M. vaccae, such as the rhamnose residue near the attachment siteof the arabinogalactan chains to the peptidoglycan backbone.

DD-M. vaccae or DD-M. vaccae-KOH (100 mg) was suspended in 1 ml of asolution of 1% periodic acid in 3% acetic acid, incubated for 1 hour atroom temperature and the solid residue recovered by centrifugation. Thisperiodic acid treatment was repeated three times. The solid residue wasrecovered by centrifugation, and incubated with 5 ml of 0.1 M sodiumborohydride for one hour at room temperature. The resulting solidresidue was recovered by centrifugation and the sodium borohydridetreatment repeated. After centrifugation, the solid residue was washedfour times with water and freeze-dried to give a yield of 62.8 mg DD-M.vaccae-periodate or 61.0 mg DD-M. vaccae-KOH-periodate.

Resuspension of DD-M. vaccae and DD-M. vaccae-KOH

DD-M. vaccae and DD-M. vaccae-KOH (11 mg each) were suspended inphosphate-buffered saline (5.5 ml). Samples were sonicated with a Virtisprobe sonicator for various times at room temperature (mini-probe, 15%output). Samples were then vortexed for sixty seconds and allowed tostand for five minutes to allow the sedimentation of large particles.The absorbance of the remaining suspension at 600 nm was measured. Asshown in FIG. 6, DD-M. vaccae-KOH (referred to in FIG. 6 as DDMV-KOH)was fully resuspended after one minute's sonication and furthersonication produced no further increase in the absorbance. After fiveminutes sonication, the resuspension of DD-M. vaccae (referred to inFIG. 6 as DDMV) was still incomplete as estimated from the absorbance ofthe suspension. These results indicate that DD-M. vaccae-KOH isconsiderably more soluble than DD-M. vaccae.

EXAMPLE 3 Effect of Immunisation with DD-M. vaccae and Derivates ofDD-M. vaccae on Asthma in Mice

The ability of DD-M. vaccae and derivatives of DD-M. vaccae to inhibitthe development of allergic immune responses was examined in a mousemodel of the asthma-like allergen specific lung disease. The severity ofthis allergic disease is reflected in the large numbers of eosinophilsthat accumulate in the lungs.

BALB/cByJ mice were given 2 μg ovalbumin in 2 mg alum adjuvant by theintraperitoneal route at time 0 and 14 days, and subsequently given 100μg ovalbumin in 50 μl phosphate buffered saline (PBS) by the intranasalroute on day 28. The mice accumulated eosinophils in their lungs asdetected by washing the airways of the anesthetized mice with saline,collecting the washings (broncheolar lavage or BAL), and counting thenumbers of eosinophils.

DD-M. vaccae derivatives were prepared as described above. Groups of 10mice were administered 200 μg of PBS, DD-M. vaccae or one of the DD-M.vaccae derivatives (Q1: DD-M. vaccae; Q2: DD-M. vaccae-KOH; Q3: DD-M.vaccae-acid; Q4: M. vaccae-periodate; Q6 and P6: DD-M.vaccae-KOH-periodate; P5: DD-M. vaccae-KOH-acid) intranasally one weekbefore intranasal challenge with ovalbumin. As shown in FIG. 3,statistically significant reductions were observed in the percentage ofeosinophils in BAL cells collected six days after challenge withovalbumin, compared to control mice. Furthermore, the data shows thatsuppression of airway eosinophilia with DD-M. vaccae-acid and DD-M.vaccae-KOH-periodate (Q3, Q6 and P6) was greater than that obtained withDD-M. vaccae (Q1). Control mice were given intranasal PBS. The data inFIG. 3 shows the mean and SEM per group of mice.

Eosinophils are blood cells that are prominent in the airways inallergic asthma. The secreted products of eosinophils contribute to theswelling and inflammation of the mucosal linings of the airways inallergic asthma. The data shown in FIG. 3 indicate that treatment withDD-M. vaccae or derivatives of DD-M. vaccae reduces the accumulation oflung eosinophils, and may be useful in reducing inflammation associatedwith eosinophilia in the airways, nasal mucosal and upper respiratorytract. Administration of DD-M. vaccae or derivatives of DD-M. vaccae maytherefore reduce the severity of asthma and diseases that involvesimilar immune abnormalities, such as allergic rhinitis.

In addition, serum samples were collected from mice immunized witheither heat-killed M. vaccae or DD-M. vaccae and the level of antibodiesto ovalbumin was measured by standard enzyme-linked immunoassay (EIA).As shown in Table 2 below, sera from mice infected with BCG had higherlevels of ovalbumin specific IgG1 than sera from PBS controls. Incontrast, mice immunized with heat-killed M. vaccae or DD-M. vaccae hadsimilar or lower levels of ovalbumin-specific IgG1. As IgG1 antibodiesare characteristic of a Th2 immune response, these results areconsistent with the suppressive effects of DD-M. vaccae on theasthma-inducing Th2 immune responses.

TABLE 2 Low Antigen-Specific IgG1 Serum Levels in Mice Immunized withHeat-killed M. vaccae or DD-M. vaccae Serum IgG1 Treatment Group MeanSEM M. vaccae i.n. 185.00 8.3 M. vaccae s.c. 113.64 8.0 DD-M. vaccaei.n. 96.00 8.1 DD-M. vaccae s.c. 110.00 4.1 BCG, Pasteur 337.00 27.2BCG, Connaught 248.00 46.1 PBS 177.14 11.4

EXAMPLE 4 Effect of DD-M. vaccae Derivates on IL-10 Production in THP-1Cells

IL-10 inhibits the cytokine production of Th₁ cells and plays a key rolein the suppression of experimentally-induced inflammatory responses inskin (Berg et al., J Exp. Med. 182:99-108, 1995). Recently, IL-10 hasbeen used successfully in two clinical trials to treat psoriaticpatients (Reich et al., J. Invest. Dermatol. 111:1235-1236, 1998 andAsadullah et al., J Clin. Invest. 101:783-794, 1998). The levels ofIL-10 produced by a human monocytic cell line (THP-1) cultured in thepresence of derivatives of DD-M. vaccae were assessed as follows.

THP-1 cells (ATCC Number TIB-202) were cultured in RPMI medium (GibcoBRL Life Technologies) supplemented with 0.5 mg/l streptomycin, 500 U/lpenicillin, 2 mg/l L-glutamine, 5×10⁻⁵ M β-mercaptoethanol and 5% fetalbovine serum (FBS). One day prior to the assay, the cells weresubcultured in fresh media at 5×10⁵ cells/ml. Cells were incubated at37° C. in humidified air containing 5% CO₂ for 24 hours and thenaspirated and washed by centrifugation with 50 ml of media. The cellswere resuspended in 5 ml of media and the cell concentration andviability determined by staining with Trypan blue (Sigma, St LouisMich.) and analysis under a hemocytometer. DD-M. vaccae derivatives(prepared as described above) in 50 μl PBS and control stimulants wereadded in triplicate to wells of a 96 well plate containing 100 μl ofmedium and appropriate dilutions were prepared. Lipopolysaccharide (LPS)(300 μg/ml; Sigma) and PBS were used as controls. To each well, 100 μlof cells were added at a concentration of 2×10⁶ cells/ml and the platesincubated at 37° C. in humidified air containing 5% CO₂ for 24 hours.The level of IL-10 in each well was determined using human IL-10 ELISAreagents (PharMingen, San Diego Calif.) according to the manufacturer'sprotocol. As shown in FIG. 4, the acid and periodate derivatives ofDD-M. vaccae were found to stimulate significant levels of IL-10production. The PBS control, DD-M. vaccae-KOH, DD-M.vaccae-KOH-periodate, and DD-M. vaccae-KOH-acid derivatives did notstimulate THP-1 cells to produce IL-10.

EXAMPLE 5 Effect of Immunizing Mice with M. vaccae, and DD-M. vaccae onTuberculosis

This example illustrates the effect of immunization with heat-killed M.vaccae or DD-M. vaccae prior to challenge with M. tuberculosis.

Mice were injected intraperitoneally with one of the followingpreparations on two occasions three weeks apart:

a) Phosphate buffered saline (PBS, control);

b) Heat-killed M. vaccae (500 μg); and

c) DD-M. vaccae (50 μg).

Three weeks after the last intraperitoneal immunization, the mice wereinfected with 5×10⁵ live H37Rv M. tuberculosis organisms. After afurther three weeks, the mice were sacrificed, and their spleenshomogenized and assayed for colony forming units (CFU) of M.tuberculosis as an indicator of severity of infection.

FIG. 5 shows data in which each point represents an individual mouse.The numbers of CFU recovered from control mice immunized with PBS alonewere taken as the baseline. All data from experimental mice wereexpressed as number of logarithms of CFU below the baseline for controlmice (or log protection). As shown in FIG. 5, mice immunized withheat-killed M. vaccae or DD-M. vaccae showed respectively a meanreduction of >1 or 0.5 logs CFU. The data demonstrates the effectivenessof immunization with M. vaccae or DD-M. vaccae and indicates that DD-M.vaccae may be developed as a vaccine against tuberculosis.

EXAMPLE 6 Compositional Analysis of DD-M. vaccae and DD-M. vaccaeDerivates

Carbohydrate compositional analysis of DD-M. vaccae and DD-M vaccaederivatives

The carbohydrate composition of DD-M. vaccae and DD-M. vaccaederivatives was determined using standard techniques. The results areshown in Table 3, wherein DDMV represents DD-M. vaccae; DDMV-KOHrepresents DD-M. vaccae-KOH; DDMV-A represents DD-M. vaccae-acid; DDMV-Irepresents DD-M. vaccae-periodate; DDMV-KOH-A represents DD-M.vaccae-KOH-acid; and DDMV-KOH-I represents DD-M. vaccae-KOH-periodate.

TABLE 3 Carbohydrate Compositional Analysis of DD-M. vaccae and DD-M.vaccae Derivatives DDMV- DDMV- DDMV- Carbohydrate DDMV KOH DDMV-A DDMV-IKOH-A KOH-I Galactosamine 26.6* 29.2 14.9 37.7 0.3 3.9 Glucosamine 3.73.6 8.7 35.6 12.2 63.2 Galactose 9.7 9.2 0.7 3.4 0.0 0.0 Glucose 56.954.8 71.1 23.0 87.5 27.5 Mannose 3.2 3.2 4.7 0.4 0.02 5.5 Fucose Notdetected Not detected Not detected Not detected Not detected Notdetected *All values in % of total carbohydrate

The results demonstrate that each of the DD-M. vaccae derivatives had adifferent carbohydrate content, as expected from the different effectsof the acid, periodate or alkali treatment of the cells. In addition,DD-M. vaccae had a marked different carbohydrate composition whencompared with the DD-M. vaccae derivatives. As expected, the amount ofgalactose in the DD-M. vaccae-acid and DD-M. vaccae-periodatederivatives was lower than in DD-M. vaccae and DD-M. vaccae-KOH. Thesevalues reflect the action of the acid and periodate in the preparationof the derivatives, cleaving the arabinogalactan sidechains from thepeptidoglycan backbone.

Nucleic Acid Analysis of DD-M. vaccae and DD-M. vaccae derivatives

Analysis by gel electrophoresis of the nucleic acid content of DD-M.vaccae and the DD-M. vaccae derivatives after treatment with ProteinaseK showed that DD-M. vaccae, DD-M. vaccae-periodate and DD-M. vaccae-KOHcontained small amounts of DNA while no. detectable nucleic acid wasobserved for DD-M. vaccae-acid.

EXAMPLE 7 Effect of DD-M. vaccae Derivates on IL-12 Production byMacrophages

The stimulation of a Th1 immune response is enhanced by the productionof interleukin-12 (IL-12) from macrophages. The ability of different M.vaccae preparations to stimulate IL-12 production was demonstrated asfollows.

A group of BALB/cByJ mice was injected intraperitoneally withthioglycolate (Difco Laboratories, Detroit Mich.). After three daysperitoneal macrophages were collected and placed in cell culture withIFN-γ (2U/ml) for four hours. The cells were washed three times in 50 mlof cold DMEM medium (Gibco Life Technologies, Gaithersburg MD)supplemented with 110mg/l pyruvate, 116 mg/l L-arginine, 36 mg/lL-asparagine, 6mg/l folic acid, 0.5 mg/l streptomycin, 500 U/lpenicillin, 2mg/l L-glutamine, 5 ×10⁻⁵ M 2-mercaptoethanol and 5% fetalbovine serum (FBS), and adjusted to a concentration of 2×10⁶ cells/ml.Various concentrations of DD-M. vaccae (referred to in FIG. 7 as R1) andDD-M. vaccae-KOH (referred to in FIG. 7 as R2), DD-M. vaccae-acid(referred to in FIG. 7 as R3), DD-M. vaccae-periodate (referred to inFIG. 7 as R4), DD-M. vaccae-KOH-acid (referred to in FIG. 7 as R5), andDD-M. vaccae-KOH-periodate (referred to in FIG. 7 as P6) were added. Toeach well, 0.1 ml of IFN-γ-treated macrophages were added at aconcentration of 2×10⁶ cells/ml. After a further 24 hours incubation at37° C., the culture supernatants were harvested and assayed for thepresence of IL-12 produced by macrophages. The level of IL-12 productionby macrophages stimulated with the DD-M. vaccae derivatives are shown inFIG. 7. The data indicates that the DD-M. vaccae derivatives stimulatedIL-12 production by macrophages at approximately the same level as DD-M.vaccae, with the exception of DD-M. vaccae-KOH-acid, which induced lessIL-12 production.

EXAMPLE 8 Effect of Immunizing Mice with Different Dosages of DD-M.vaccae Derivates

This example illustrates the effect of immunization with differentdosages of DD-M. vaccae derivatives on the development of an allergicimmune response in the lungs. This was demonstrated in a mouse model ofthe asthma-like allergen specific lung disease. The severity of thisallergic disease is reflected in the large numbers of eosinophils thataccumulate in the lungs.

BALB/cByJ female mice were sensitized to OVA by intraperitonealinjection of 200 μl of an emulsion containing 10 μg OVA and 1 mg Alumadjuvant on days 0 and 7. On days 14 and 21, mice were anesthetized andvaccinated intranasally or intradermally with 200 μg of DD-M.vaccae-acid or PBS. On days 28 and 32, mice were anesthetized andchallenged intranasally with 100 μg OVA. Mice were sacrificed on day 35and bronchoalveolar lavage (BAL) performed using PBS. BAL samples wereanalyzed by flow cytometry to determine the eosinophil content (%eosinophils). Total BAL eosinophil numbers were obtained by multiplyingthe percentage eosinophil value with the total number of leukocytesobtained, with the latter value being determined using a hemacytometer.

As can be seen in FIG. 8, DD-M. vaccae-acid caused a statisticallysignificant, dosage-dependent suppression of airway eosinophilia (%eosinophils), with increasing levels of suppression being observed withincreasing dosages of DD-M. vaccae-acid.

EXAMPLE 9 Effects of the Route of Immunization of Mice with Derivates ofDD-M. vaccae

This example illustrates the effect of different routes of immunizationwith DD-M. vaccae derivatives on the suppression of eosinophilia in thelung in a mouse model of the asthma-like allergen specific lung disease.

BALB/cByJ female mice were sensitized to OVA by intraperitonealinjection of 200 μl of an emulsion containing 10 μg OVA and 1 mg Alumadjuvant on days 0 and 7. On days 14 and 21, mice were anesthetized andvaccinated intranasally or intradermally with 200 μg of DD-M.vaccae-acid or PBS. On days 28 and 32, mice were anesthetized andchallenged intranasally with 100 μg OVA. Mice were sacrificed on day 35and bronchoalveolar lavage (BAL) performed using PBS. BAL samples wereanalyzed by flow cytometry to determine the eosinophil content (%eosinophils). Total BAL eosinophil numbers were obtained by multiplyingthe percentage eosinophil value with the total number of leukocytesobtained, with the latter value being determined using a hemacytometer.

As shown in FIG. 9, statistically significant reductions were observedin the percentage of eosinophils in BAL cells collected six days afterchallenge with ovalbumin from mice immunized intranasally with DD-M.vaccae-acid, compared to control mice. Furthermore, the data shows thatsuppression of airway eosinophilia with DD-M. vaccae-acid administeredintranasally was greater than that when mice were immunizedintradermally. Control mice were given intranasal PBS. The data in FIG.9 shows the mean and SEM per group of mice.

EXAMPLE 10 Preparation and Compositional Analisys of Delipidated andDeglycolipidated M. tuberculosis (DD-M. tuberculosis) and M. smegmatis(DD-M. smegmatis)

M. tuberculosis and M. smegmatis Culture Filtrate

Cultures of Mycobacterium smegmatis (M. smegmatis, ATCC Number 27199)were grown as described in Example 1 for M. vaccae in Medium 90 with 1%added glucose. After incubation at 37° C. for 5 days, the cells wereharvested by centrifugation and the culture filtrate removed. Thebacterial pellet was resuspended in phosphate buffered saline at aconcentration of 10 mg/ml, equivalent to 10¹⁰ M. smegmatis organisms perml. The cell suspension was then autoclaved for 15 min at 120° C. Theculture filtrate was passaged through a 0.45 μm filter into sterilebottles.

Cultures of M. tuberculosis strain H37Rv (ATCC Number 27294) were grownat 37° C. in GAS medium (0.3 g Bactocasitone (Difco Laboratories,Detroit MI), 0.05 g ferric ammonium citrate, 4 g K₂HPO₄, 2 g citricacid, 1 g L-alanine, 1.2 g MgCl₂.6H₂O, 0.6 g K₂SO₄, 2 g NH₄Cl, 1.8 mlNaOH (10 N), 5 ml glycerol, pH 7.0) for five days. Harvesting andfurther treatment of cells are as described above for M. smegmatiscells.

Preparation of Delipidated and Deglycolipidated M. tuberculosis (DD-M.tuberculosis) and Delipidated and Deglycolipidated M. smegmatis (DD-M.smegmatis) and Compositional Analysis

To prepare delipidated and deglycolipidated M. tuberculosis (DD-M.tuberculosis) and M. smegmatis (DD-M. smegmatis), autoclaved M.tuberculosis and M. smegmatis were pelleted by centrifugation, thepellet washed with water and collected again by centrifugation, andfreeze-dried. An aliquot of this freeze-dried M. tuberculosis and M.smegmatis was set aside and referred to as lyophilized M. tuberculosisand M. smegmatis, respectively. When used in experiments the lyophilizedmaterial was resuspended in PBS to the desired concentration.

Delipidated and deglycolipidated M. tuberculosis and M. smegmatis wereprepared as described in Example 1 for the preparation of DD-M. vaccae.For bioassay, the freeze-dried DD-M. tuberculosis and DD-M. smegmatiswere resuspended in phosphate-buffered saline (PBS) by sonication, andsterilized by autoclaving.

The compositional analyses of DD-M. tuberculosis and DD-M. smegmatis arepresented in Table 4 and Table 5. Major differences are seen in somecomponents of the monosaccharide composition of DD-M. tuberculosis andDD-M. smegmatis compared with the monosaccharide composition of DD-M.vaccae. The data presented in Table 4 show that DD-M. tuberculosis andDD-M. smegmatis contain 1.3% and 0.0 mol % glucose, respectively,compared with 28.1 mol % for DD-M. vaccae.

The amino acid composition of DD-M. tuberculosis and DD-M. smegmatis ispresented in Table 5. DD-M. tuberculosis contains 6537.9 nmoles/mg aminoacids, or approximately 78.5% w/w, and DD-M. smegmatis contains 6007.7nmoles/mg amino acids, which is approximately 72.1% w/w protein. Whencompared with the amino acid analysis of DD-M. vaccae given in Table 1,DD-M. tuberculosis and DD-M. smegmatis contain more total % protein thanDD-M. vaccae (55.1%).

TABLE 4 Monosaccharide Composition of DD-M. tuberculosis and DD-M.smegmatis M. tuberculosis M. smegmatis Monosaccharide wt % mol % wt %mol % Inositol 0.0 0.0 0.0 0.0 Glycerol 9.5 9.7 15.2 15.5 Arabinose 69.371.4 69.3 70.0 Xylose ND* ND 3.9 4.0 Mannose 3.5 3.0 2.2 1.9 Glucose 1.51.3 0.0 0.0 Galactose 12.4 10.7 9.4 8.0 *Not done

TABLE 5 Amino Acid Composition of DD-M. tuberculosis and DD-M. smegmatisM. tuberculosis M. smegmatis Total Protein Total % Total Protein Total %Amino acid nmoles/mg protein nmoles/mg protein ASP 592.5 9.1 557.0 9.3THR 348.1 5.3 300.5 5.0 SER 218.6 3.3 252.6 4.2 GLU 815.7 12.5 664.911.1 PRO 342.0 5.2 451.9 7.5 GLY 642.9 9.8 564.7 9.4 ALA 927.9 14.2875.1 14.6 CYS 31.8 0.5 20.9 0.3 VAL 509.7 7.8 434.8 7.2 MET 122.6 1.9113.1 1.9 ILE 309.9 4.7 243.5 4.1 LEU 542.5 8.3 490.8 8.2 TYR 116.0 1.8108.3 1.8 PHE 198.9 3.0 193.3 3.2 HIS 126.1 1.9 117.2 2.0 LYS 272.1 4.2247.8 4.1 ARG 421.0 6.4 371.7 6.2

EXAMPLE 11 Effect of Immunization with DD-M. tuberculosis and DD-M.smegmatis on Asthma in Mice

The ability of DD-M. tuberculosis and DD-M. smegmatis to inhibit thedevelopment of allergic immune responses was examined in a mouse modelof the asthma-like allergen specific lung disease, as described above inExample 8. The results illustrates the effect of immunization with DD-M.tuberculosis and DD-M. smegmatis on the suppression of eosinophilia inthe lung, illustrating their immune modulating properties.

BALB/cByJ female mice were sensitized to OVA by intraperitonealinjection of 200 μl of an emulsion containing 10 μg OVA and 1 mg Alumadjuvant on days 0 and 7. On days 14 and 21, mice were anesthetized andvaccinated intranasally or intradermally with 200 μg of DD-M. vaccae,DD-M. tuberculosis, DD-M. smegmatis or PBS. On days 28 and 32, mice wereanesthetized and challenged intranasally with 100 μg OVA. Mice weresacrificed on day 35 and bronchoalveolar lavage (BAL) performed usingPBS. BAL samples were analyzed by flow cytometry to determine theeosinophil content (% eosinophils). Total BAL eosinophil numbers wereobtained by multiplying the percentage eosinophil value with the totalnumber of leukocytes obtained, with the latter value being determinedusing a hemacytometer.

The data shown in FIG. 10 indicate that treatment with DD-M.tuberculosis and DD-M. smegmatis reduces the accumulation of lungeosinophils similar to the reduction following immunization with DD-M.vaccae, and that DD-M. tuberculosis and DD-M. smegmatis may be useful inreducing inflammation associated with eosinophilia in the airways, nasalmucosal and upper respiratory tract. Administration of DD-M.tuberculosis and DD-M smegmatis may therefore reduce the severity ofasthma and diseases that involve similar immune abnormalities, such asallergic rhinitis.

EXAMPLE 12 Effect of DD-M. vaccae on Production of IL-10, TNF-Alpha andINF-Gamma in Human Peripheral Blood Mononuclear Cells

This example describes studies on the ability of DD-M. vaccae tostimulate cytokine production in human peripheral blood mononuclearcells (PBMC).

Human blood was separated into PBMC and non-adherent cells, and thecytokine production of each fraction determined after stimulation withDD-M. vaccae as follows. Blood was diluted with an equal volume ofsaline and 15-20 ml was layered onto 10 ml Ficoll (Gibco BRL LifeTechnologies, Gaithersburg, Md.). The lymphocyte layer was removed aftercentrifugation at 1,800 rpm for 20 min, washed three times in RPMImedium (Gibco BRL) and counted using Trypan blue. Cells were resuspendedin RPMI containing 5% heat-inactivated autologous serum at aconcentration of 2×10⁶ per ml. The cell sample was divided to preparenon-adherent cells.

Non-adherent cells were prepared by incubating 20 ml of the lymphocytesin RPMI supplemented with serum (as above) for one hour in a humidifiedatmosphere containing 5% CO₂. The non-adherent cells were transferred toa fresh flask and the incubation repeated once more. The non-adherentcells were removed, counted and resuspended at a concentration of 2×10⁶per ml in supplemented RPMI medium. Serial dilutions of DD-M. vaccaewere prepared starting at 200 μg/ml and added to 100 μl medium(supplemented RPMI) in a 96-well plate. PBMC and non-adherent cells wereadded to the wells (100 μl) and the plates incubated at 37° C. for 48hours in a humidified atmosphere containing 5% CO₂. A 150 μl aliquot wasremoved from each well to determine the amount of cytokine produced bythe different cells after stimulation with DD-M. vaccae.

DD-M. vaccae stimulated PBMC to secrete TNF-α and IL-10 (FIGS. 11 and12A, respectively), but stimulated the non-adherent cells to produceIFN-γ (FIG. 12B). These data suggest that IFN-γ production in DD-M.vaccae-stimulated PBMC is repressed by the simultaneous secretion ofIL-10.

EXAMPLE 13 Activation of T Cells by Heart-killed M.vaccae and DD-M.vaccae

The ability of heat-killed M. vaccae and DD-M. vaccae to activate humanT cells and Natural Killer (NK) cells was examined as follows.

Human peripheral blood mononuclear cells (PBMC) at a concentration of5×10⁶ cells per ml were cultured with 20 ug/ml of either heat-killed M.vaccae or DD-M. vaccae for 24 hours. Control cells were cultured withmedia alone. Cultured cells were then stained with monoclonal antibodiesagainst CD56 (a marker for NK cells), αβT cells, or γδT cells incombination with monoclonal antibody against CD69, a molecule expressedby activated cells. The cells were then analyzed by flow cytometry. Thepercentage of cells expressing CD69 are provided in Table 6.

TABLE 6 Activation of Human T Cells and NK Cells by Heat-Killed M.vaccae and DD-M. vaccae αβT cells γδT cells NK cells Control 3.8 6.2 4.8Heat-killed M. vaccae 8.3 10.2 40.3 DD-M. vaccae 10.1 17.5 49.9

These results indicate that heat-killed M. vaccae and DD-M. vaccaeactivate both αβ and γδT cells, as well as NK cells.

Recent studies by Holt and Sly (Nature Medicine, 1999, 5:1127-1128)indicate that, in asthma, γδT cells are important in maintaining normalairway responsiveness and downregulate airway responsiveness to allergenchallenge, possibly by controlling the “repair” response of the airwayepithelium to γδT cells cell-mediated damage. Since M. vaccae and DD-M.vaccae are able to activate γδT cells, they are likely to effective inrestoring a normal epithelium in diseased areas of the body where γδTcells are found, such as airways, lungs, skin and gut.

Although the present invention has been described in some detail by wayof illustration and example for purposes of clarity of understanding,changes and modifications can be carried out without departing from thescope of the invention which is intended to be limited only by the scopeof the appended claims.

We claim:
 1. A composition comprising delipidated and deglycolipidatedMycobacterium vaccae cells that have been treated by acid hydrolysis. 2.The composition of claim 1, wherein the delipidated and deglycolipidatedMicrobacterium vaccae cells that have been treated by acid hydrolysiscontain galactose in an amount less than 9.7% of total carbohydrate. 3.The composition of claim 1, wherein the delipidated and deglycolipidatedMicrobacterium vaccae cells that have been treated by acid hydrolysiscontain glucosamine in an amount greater than 3.7% of totalcarbohydrate.
 4. The composition of claim 1, wherein the delipidated anddeglycolipidated Microbacterium vaccae cells that have been treated byacid hydrolysis contain galactosamine in an amount less than 26.6% oftotal carbohydrate.
 5. The composition of claim 1, wherein thedelipidated and deglycolipidated Microbacterium vaccae cells that havebeen treated by acid hydrolysis contain glucose in an amount greaterthan 56.9% of total carbohydrate.
 6. The composition of claim 1, whereinthe delipidated and deglycolipidated Microbacterium vaccae cells thathave been treated by acid hydrolysis contain mannose in an amountgreater than 3.2% of total carbohydrate.
 7. The composition of claim 1,additionally comprising an adjuvant.
 8. A composition comprisingdelipidated and deglycolipidated Mycobacterium vaccae cells that havebeen treated with periodic acid.
 9. The composition of claim 8, whereinthe delipidated and deglycolipidated Microbacterium vaccae cells thathave been treated with periodic acid contain galactose in an amount lessthan 9.7% of total carbohydrate.
 10. The composition of claim 8, whereinthe delipidated and deglycolipidated Microbacterium vaccae cells thathave been treated with periodic acid contain glucosamine in an amountgreater than 3.7% of total carbohydrate.
 11. The composition of claim 8,additionally comprising an adjuvant.
 12. A composition comprisingdelipidated and deglycolipidated Mycobacterium vaccae cells that havebeen treated by alkaline hydrolysis and by acid hydrolysis.
 13. Thecomposition of claim 12, wherein the delipidated and deglycolipidatedMicrobacterium vaccae cells that have been treated by alkalinehydrolysis and by acid hydrolysis contain galactose in an amount lessthan 9.7% of total carbohydrate.
 14. The composition of claim 12,wherein the delipidated and deglycolipidated Microbacterium vaccae cellsthat have been treated by alkaline hydrolysis and by acid hydrolysiscontain glucosamine in an amount greater than 3.7% of totalcarbohydrate.
 15. The composition of claim 12, additionally comprisingan adjuvant.
 16. A composition comprising delipidated anddeglycolipidated Mycobacterium vaccae cells that have been treated byalkaline hydrolysis and treated with periodic acid.
 17. The compositionof claim 16, wherein the delipidated and deglycolipidated Microbacteriumvaccae cells that have been treated by alkaline hydrolysis and treatedwith periodic acid contain galactose in an amount less than 9.7% oftotal carbohydrate.
 18. The composition of claim 16, wherein thedelipidated and deglycolipidated Microbacterium vaccae cells that havebeen treated by alkaline hydrolysis and treated with periodic acidcontain glucosamine in an amount greater than 3.7% of totalcarbohydrate.
 19. The composition of claim 18, additionally comprisingan adjuvant.